Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/01—Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
  • C11D1/66—Non-ionic compounds
  • C11D1/662—Carbohydrates or derivatives
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/20—Organic compounds containing oxygen
  • C11D3/2096—Heterocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/12—Disaccharides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters

Definitions

• the present invention relates to an improved process for the production of sophorolactone, to a sophorolactone composition in the form of a solid or paste obtainable by the method and to formulations comprising sophorolactone obtained by the method.
• the present invention relates to processes for the manufacturing of derivatives of the sophorolactone, to the sophorolactone derivatives and to uses thereof.
• Sophorolipids formerly known as sophorosides, are glycolipid biosurfactants produced by yeast strains such as Starmerella (Candida) bombicola, Candida apicola, Candida bogoriensis, Candida batistae and Wickerhamilella domercqiae. They are composed of a disaccharide moiety linked to one hydroxyl group of one w or (w-l)-hydroxy fatty acid that is saturated or monounsaturated.
• the sugar moiety i.e. sophorose or 2-0- glucopyranosyl— D-glucopyranose, may further show mono-or diacetylation at the 6' and 6" positions.
• sophorolipids are considered as being a mixture of the compounds presented by the formula I, representing sophorolipids in lactone form, and by formula II, representing sophorolipids in acid form, in which 1.
• R' and R" represent hydrogen or an acetyl group, or 2.
• R' is an acetyl group and R"" a hydrogen group, or 3.
• R' is hydrogen and R" is an acetyl group, or 4. both R' and R" are hydrogen, or 5. both R' and R" are acetyl groups.
• the molecules of formula I are lactonic sophorolipids, also called sophorolactones.
• the molecules of formula II are open ring sophorolipids, also called sophorolipids in acid form.
• Sophorolipids are typically produced by fermentation processes wherein a glycolipid producing micro-organism is fed with a sugar supply and a substrate under appropriate fermentation conditions for the production of the sought sophorolipids.
• This process has the disadvantage that a group of numerous homologs is formed. Moreover the formation ratio of these homologs varies as a function of their substrate, e.g . a hydrocarbon source, as well as the fermentation conditions. The production of a product having a given ratio using a fermentation process is difficult. This also hampers product development as properties and function of the sophorolipid varies with ratios of the homologs.
• sophorolipids are chemically unstable and are very easily hydrolysed by heating or prolonged storage close to neutrality or even at ambient temperature under slightly alkaline conditions, which leads to the obtaining of the completely de-acetylated acid form. It is therefore extremely difficulty by fermentation or chemistry to obtain a single product and a fortiori an acetylated product.
• Processes for the isolation of one of the main forms (acid or lactone) from sophorolipids have been investigated. The processes thus far described require extraction by alcohol, e.g. EP 209783, a process which is both long and expensive.
• sophorolipids as biosurfactants. It is undesirable that the product contains traces of organic solvents, resulting from the separation process.
• the lactone form of sophorolipids has been documented to be the most active sophorolipid type, both with regard to lowering surface tension and to antimicrobial activity.
• sophorolactone that allows the production of derivatives such as w or (w-l)-hydroxy fatty acids and/or sophorose on an economically relevant scale.
• sophorolactone compositions with improved properties, such as hydrolytic stability, increased ease of handling. It is also an object of the invention to provide a more economically friendly access to some uses of sophorolactone and its derivatives.
• the present invention thereto provides an improved process for the selective production of sophorolactone in absence of an organic solvent comprising the steps of:
• the inventors have found that less acid sophorolipids are formed when the manufacturing process is split up over a step using a preculture without oil and a fermentation step using an oily substrate; and keeping fatty acid levels below a pre-determined level in the fermentation broth.
• the method is also applicable to substrates other than oils, said other substrates including shielded esters, for instance as described in EP1953237, and alcohols, such as described in Brakemeier A. et al, Biotechnology letters, vol. 17, No. 11, nov. 1995, P1183-1188 or Fleurackers S. J. J. et al, Eur. J. Lipid Sci. Technol. 2010 ( 112) : 655-662.
• the decreased formation of acid sophorolipids has two advantages; first the envisaged lactone content will be higher and downstream processing easier. Secondly due to the lower content of acid sophorolipids, which foam more than sophorolactones, foam control will be easier. Hence the addition of antifoaming agents, which are difficult to remove later on, can be avoided.
• the fermenter capacity can thus be made maximal use of and a crude sophorolactone composition with low residual substrate content will result, allowing for lactone isolation. This provides access to sophorolactone in solid form, such as a powder, with improved hydrolytic stability due to the absence of water.
• a process according to an embodiment of the invention has the advantage that it is straightforward, simple to execute and results in a less complex sophorolipid mixture than prior art procedures. It has for effect that high yields of sophorolactone are obtained. Suppression of the acid levels has for effect that no emulsion is formed and the lactone form will not be solubilized.
• aqueous process of the invention does not require the use of organic solvents to obtain sophorolactone.
• the resulting products are free of traces of organic solvent.
• the process comprising the steps of:
• water is removed from the crystals prior to drying by melting the crystals to a temperature between 35°C and 55°C, thereby providing a lower layer comprising sophorolactone and an upper layer of supernatant water; separating the lower layer from the upper layer to obtain a sophorolactone composition with increased active matter content.
• the drying is by spray drying, drum drying, convection drying, thin film evaporation, vacuum drying, flaking, extruding or casting.
• the sophorolactone composition separated off the reaction mixture is in the form of an aqueous sophorolipid mixture comprising at least 30 wt%, preferably at least 70 wt%; more preferably at least 80 wt% sophorolactone, expressed on the dry matter content of the aqueous sophorolipid mixture.
• the sophorolactone is comprised in a composition comprising at most 60 weight percent water.
• the fatty acid levels are suppressed by adjusting the level of substrate fed to the metabolizing cells.
• the sugar and substrate are fed to the pre-cultivated cells in a fed batch mode.
• the fatty acid level of the reaction mixture is suppressed below 10 g/l, preferably below 8 g/l, more preferably between 1-5 g/l, expressed in weight of acid per liter of reaction mixture.
• the substrate comprises corn steep liquor.
• the pre-cultivation is carried out in the absence of an oil.
• the substrate is a triglyceride; preferably a triglyceride of saturated or unsaturated fatty acids with 16 or 18 carbon atoms; more preferably a vegetable oil; most preferably rapeseed oil.
• the substrate is isostearic acid or isostearic alcohol, preferably isostearyl alcohol.
• the substrate a shielded ester of formula H-(CH 2 )n-0-CO-(CH 2 )m-COOH with an carbon chain length of 16 to 18.
• the substrate comprises a carbon chain length lower than C14, preferably between C8 and C14
• the fermentation medium comprises cells of a Candida species modified to improve the conversion of said substrate with short carbon chain length into sophorolactone.
• the Candida is Candida bombicola mutant strain M 18, M30 or M33; preferably Candida bombicola M30.
• the process additionally comprising the step of filtering the sophorolactone separated off the reaction mixture by depth filtration, more preferably by depth filtration at a temperature between 40 °C and 90 °C and pressure of between 0.5 bar and 2.5 bar; most preferably by depth filtration at a temperature between 60 °C and 70 °C and pressure between 0.5 and 2.0 bar.
• the fermentation is executed in a vessel at an overpressure of between 0-1.5 bar, preferably at 0.5-0.6 bar. This allows control of foam formation.
• the present invention provides a sophorolactone composition in the form of a solid or paste, obtainable by a method according to an embodiment of the invention, consisting of 60-70 wt% sophorolactone, 0-30 wt% sophorolipid acid, 0-1 wt% residual substrate including less than 0.2 wt% fatty acid, and remainder water.
• residual substrate it is meant the sum of the non-metabolised substrate and the metabolites other than sophorolipids thereof, i.e. triglycerides and fatty acids, fatty alcohol and fatty acid, methylesters and fatty acids or fatty acids as such .
• the sophorolactone composition obtainable by a method according to the invention comprises at least 60 weight percent sophorolactone and at most 40 weight percent water.
• the sophorolactone composition consists of 70-100 wt% sophorolipid lactone, 0-1 wt% sophorolipid acid, preferably 0-0.5% sophorolipid acid, less than 0.1 % residual substrate including fatty acid, and remainder water.
• the present invention provides a formulation obtained by dissolving the sophorolactone composition according to an embodiment of the invention in a solvent with a polarity parameter delta P between 5 and 9.5 and a hydrogen bonding parameter delta H between 8 and 15.
• the solvent in a formulation according to an embodiment of the invention is selected from the list of phenoxyethanol, benzylalcohol, phenetyl alcohol, hydrocinnamylalcohol, tetrahydrofurfuryl alcohol, dimethylisosorbide, methyl salicylate and eugenol.
• the invention provides a dry solid product for laundry or dishwashing, comprising a sophorolactone composition consisting of 70-100 wt% sophorolipid lactone, 0-lwt% sophorolipid acid, less than 0.1% residual substrate and remainder water; wherein the pH of the product is 8.0-14.0, preferably 8.5-11.50, as measured on a 1% solution of the product in water.
• a process for the preparation of partially hydrolysed sophorolactone comprising the steps of:
• sophorolactone -hydrolysing at least part of the sophorolactone, either by adding to the sophorolactone 0.5 to 1 equivalents of a base or by heating the sophorolactone for more than 4 hours, preferably more than 24 hours, to a temperature between 70-
• Suitable bases for use are potassium hydroxide, sodium hydroxide, magnesium hydroxide, ammonium hydroxide, monoethanolamine, guanidine or guanidinium hydroxide.
• the foam behavior and desired solubility of the product can be tuned by the amount of base equivalents used for the hydrolysation.
• the invention provides a method for the preparation of a ⁇ hydroxy fatty acid of formula CH20H-CH2-(CH2)n-COOH or a ⁇ -l hydroxy fatty acid of formula
• This preparation method is advantageous as it provides sophorose. This is in contrast to an acid hydrolysis whereby the sophorose is lost.
• the hydroxy fatty acid is derived from isostearine acid.
• the separation of the sophorolipid acid into a sugar moiety and ⁇ and ⁇ -l hydroxy fatty acid moiety is by enzymatic reaction, preferably in the presence of endoglycoceramidase II recombinant (E.C. 3.2.1.123). More preferably sophorolipid acids free of esteric bonds are used as starting material for the enzymatic reaction. This ensures ready access of the enzyme to the substrate. Alkaline hydrolysis of the sophorolactone followed with a suitable purification process such as crystallization in an acid pH would hydrolyze all esteric bonds but leave the glycosidic bonds intact.
• a 1% aqueous sophorolactone solution expressed in weight of the sophorolactone by total weight of the solution, at a pH of 5,5 combined with 1.5 units of the endoglycoceramidase II enzyme is then reacted for at least 16 hours, preferably at least 24 hours, at a temperature of 37°C.
• the invention provides hydroxy fatty acid obtained by a method according to an embodiment of the invention, characterized by the virtual absence of non hydroxylated fatty acids, meaning in a concentration below 0.1%.
• Figure 1 is a graphic representation of the results of solubility experiments of sophorolactones, expressed in Hansen solubility parameters delta H and delta P.
• Figure 2 is a graphic representation of the oil feeding rate corresponding with the experiment as described in Example 1.
• the speed of addition of oil expressed in g/h
• the amount of oil expressed in g
• the duration of the process is displayed, expressed in hours. Residual oil levels are monitored and kept below a pre-determined level of 8 g/l.
• Figure 3 is a graphic representation of a chromatogram recorded for the experiment described in Example 8 (1 st wash, 1% in ethanol, 100 ⁇ injection volume).
• Figure 4 is a graphic representation of a chromatogram recorded for the experiment described in Example 9 (sophorolactone with C18 carbon chain, 1% in tetrahydrofuran, 5 ⁇ injection volume).
• Figure 5 is a graphic representation of a chromatogram recorded for the experiment described in Example 13. The chromatogram was recorded on a mixture of water-soluble sophorolipids, saturated with lactonic sophorolipids.
• Figure 6 is a graphic representation of a chromatogram recorded for the experiment described in Example 13. The chromatogram was recorded on a mixture of sophorolipids out of which solid lactonic sophorolipids could be obtained through dispersion in water.
• Figure 7 is a graphic representation of a chromatogram recorded for the experiment described in Example 16 (bottom phase, 1% in ethanol, 20 ⁇ injection volume) .
• Figure 8 is graphic representation of a chromatogram recorded on a non-aqueous sophorolactone solution prior to storage (black curve) and after storage (grey curve) at 4 °C for 2 months. Both curves practically overlap.
• the solution is storage stable as demonstrated by the virtual absence of a peak at a retention time of 30,5 min. in the grey overlay graph which would demonstrate the presence of acid sophorolipid, derived from hydrolyzed lactones.
• Figure 9 is graphic representation of a chromatogram recorded on the same sample as for Figure 5, after 2 months of storage at 40°C. Both curves practically overlap.
• the solution is storage stable as demonstrated by the virtual absence of a peak at a retention time of 30,5 min. in the grey overlay graph which would demonstrate the presence of acid sophorolipid, derived from hydrolyzed lactones.
• the present invention concerns an improved method for producing sophorolactone, sophorolactone in solid form obtained with an improved degree of purity and uses for this sophorolactone.
• the access to sophorolactone provided by the invention further allows improvements in the production of sophorolactone derivatives.
• the invention provides a method for producing at least partially hydrolysed sophorolactone, ⁇ and ⁇ -l hydroxy fatty acids and sophorose derived from sophorolactone.
• a compartment refers to one or more than one compartment.
• the value to which the modifier "about” refers is itself also specifically disclosed.
• sophorolipid refers to the non-acetylated sophorolipid as well as to its acetylated derivative.
• Candida species capable of producing sophorolactone are Candida bombicola and/or Candida apicola; most preferably Candida bombicola.
• the process is split-up over a step wherein Candida species are cultivated in the absence of an oil or oily substrate.
• This step will be named the pre-cultivation.
• pre-culture it is meant herein, the stage of the synthesis process preceding the fermentation steps. This preculture stage yields the required Candida biomass for appropriate inoculation of the full fermentation volume.
• micro-organisms can be displayed in a biomass growth curve. It typically displays a part of exponential growth, the so-called log phase or phase of exponential growth, followed by a stationary phase, in which the size of a population of microorganisms remains constant, even though some cells continue to divide and others begin to die. As the population grows, microorganisms consume nutrients and produce waste products. When the nutrient supply is depleted, the growth rate enters a stationary phase in which the number of viable cells remain the same.
• cells of the selected Candida species are pre-cultivated until a stationary growth phase is obtained. This deviates from the prior art wherein a pre- culture is typically obtained from cell growth up to the stage of experimental growth. The inventors found that a pre-culture of cells grown until the stationary growth phase was reached was advantageous to increase sophorolactone yield.
• the pre-cultivated cells thus obtained are then used in a culture wherein the pre-cultivated cells are brought in contact with at least one fermentable sugar and at least one fermentable substrate.
• the cells are provided with adequate supply of sugar and substrate.
• Suitable sugars for use in the invention are glucose, sucrose and raffinose.
• the sugar is glucose either in solid or in liquid form and glucose levels are maintained in excess of 20 g/l by intermittent glucose addition.
• substrates that can be converted into sophorolipids, either by direct glycosidic coupling of a glucose unit to the (hydroxylated) substrate as such or by glycosidation after biochemical transformation of the substrate (e.g. hydroxylation) by the Candida species.
• the monoglycoside is subsequently converted into a sophorolipid by coupling to a second glucose unit.
• Suitable substrates for use in the invention are alkanes, fatty alcohols, fatty acids and their esters, including triglycerides and shielded esters. These substrates can be linear or branched, saturated, mono- or polyunsaturated with a carbon chain of 5 to 18 carbon atoms, preferably 12 to 18 carbon atoms, more preferably 15 to 18 carbon atoms.
• the substrate is a triglyceride; preferably a triglyceride of saturated or unsaturated fatty acids with 16 or 18 carbon atoms; more preferably a vegetable oil; most preferably rapeseed oil.
• the substrate is isostearic acid or isostearic alcohol, preferably isostearyl alcohol.
• aqueous medium aqueous medium
• an organic solvent to harvest the sophorolactone is not required.
• the production and isolation of sophorolactone may be executed in aqueous media, without organic solvents.
• a shielded ester designates a compound represented by formula III, or a salt or a methyl, ethyl or glycerol ester of the compound of formula III which contains at least one cleavable bond
• Y is -0-, -S-, -NH-, a mono or di unsaturated bond
• X and Z may be the same or different and are chosen from the group of a -CO- group or a -CH n (CH 2 )m- group which may be straight or branched, with m having a value between 0 and 4 and n between 0 and 2,
• R 1 and R 2 each are aliphatic hydrocarbon chains which may be the same or different chain, which may be branched or unbranched, may contain one of more unsaturated bonds and may contain one or more substituents,
• R 10 and R 20 may be the same or different and are chosen from the group of -H, - CH 3 , -CHO, -CH 2 OH, -COOH, -CHS, -CH 2 SH, -CN or -CH 2 NR 3 P , R 3 being an aliphatic hydrocarbon chain or H, p being 1 or 2,
• -R ⁇ -R ⁇ X is a lipophilic moiety of the glycolipid
• the number of atoms in the chain determining the chain length of formula III is between 14 and 22, preferably between 16 and 18 and in that the at least one cleavable bond is broken to remove the elongator part from the glycolipid to obtain a short chained glycolipid.
• the compound of formula III is a so called shielded ester, wherein the hydrocarbon chain which determines the chain length of each of R ⁇ -R ⁇ X- and -Y-Z-R 2 -R 20 contains at least 3 carbon atoms.
• the R ⁇ -R 1 - and/or -R 2 -R 20 part of the shielded ester may contain unsaturated carbon-carbon bonds.
• a shielded ester designates a compound represented by formula III, wherein the functional group -X-Y-Z- is spaced apart from the end part of formula I by 3 or more carbon atoms.
• Such shielded, cleavable esters may be synthesised chemically or enzymatically using methods well known to the person skilled in the art.
• the functional group -X-Y-Z- is sufficiently far away from the terminal part of the molecule, so that its accessibility to extra-cellular enzymes is minimal.
• terminal esters - i.e. methyl-, ethyl- esters and glycerides - as substrates for sophorolipid synthesis has been well documented, these esters are readily hydrolysed by extracellular enzymes into C16-18 carboxylic acids.
• Terminal esters will mainly yield C18 sophorolipids, which can be converted into shorter chained sophorolipids in case they contain one or more unsaturated carbon-carbon bonds. This can for instance be achieved by subjecting the C18 sophorolipid to ozonolysis, removing any salts and dicarboxylic acids and obtaining sophorolipids with a chain length of 9 carbon atoms; as is described in EP1953237.
• Examples of shielded esters suitable for use in the present invention are provided in Table 1 below, n is the number of carbon atoms in the chain length determining part of the alcohol and m the number or carbon atoms in the chain length determining part of the carboxylic acid minus two.
• the substrate is a shielded ester of formula H-(CH 2 )n-0-CO-(CH 2 )m-COOH with an apparent chain length of 16 to 18, i.e. with 15 to 17 carbon atoms.
• the substrate comprises a carbon chain length lower than C14, preferably between C8 and C14
• the fermentation medium comprises cells of a Candida species modified to improve the conversion of said substrate with short carbon chain length into sophorolactone.
• Candida species are for instance the Candida bombicola mutant strain M 18, M30 or M33; preferably Candida bombicola M30.
• Use of these modified species has for effect that the sophorolactones will be produced with a corresponding chain length in their fatty acid moiety at an improved yield. Yield will be improved as these species have been genetically modified so that the beta-oxidation pathway is blocked.
• a side reaction wherein a substrate comprising a carbon chain length below C14 is metabolized rather than converted into a sophorolipid is blocked.
• the pre-cultivated cells are fermented in a fed-batch set-up.
• the sugar and substrate are fed to the pre-cultivated cells in fed batch mode.
• Use of a fed-batch fermentation as production technique has the advantage that the nutritional environment required for fermentation can be kept approximately constant during the course of the batch.
• the production of by-products that are generally related to the presence of high concentrations of substrate can be avoided by limiting its quantity to the amounts that are required solely for the production of the sophorolipid.
• the substrate comprises corn steep liquor or CSL.
• Corn steep liquor is known as a source of lactic acid. It is a commercially available product. It is a viscous yellowish or dens brown liquid or powder obtained by concentration of corn steep water.
• the dry matter content is usually 45-55%, preferably 48-52%.
• the pH is in the range of 3-5, preferably 3.5-4.5.
• the protein content (Dry Matter) is typically 30-50%, preferably 35-45%.
• the growth medium comprises 50-60 g/l glucose.
• corn steep liquor is in the form of a powder.
• Table 1 Examples of preferred shielded esters of formula H-(CH 2 )n-0-CO-(CH 2 )m-COOH n monoAlcohol m diAcid Total *
• the conditions comprise selection of a suitable temperature and suitable oxygen supply.
• a suitable temperature is in the range of 24°C-30°C, more preferably about 25°C.
• Suitable oxygen supply can be pure oxygen, oxygen enriched air or preferably air, the latter introduced in the fermenter at an aeration rate from 0.05-1.5 vvm, preferably starting at 0.5- 1 vvm and reducing the aeration in case of risk for overfoaming to 0.05- 0.5 vvm.
• sophorolipid composition comprising sophorolipids in acid form and sophorolipids in lactone form.
• the inventors have found that it is particularly advantageous to suppress the formation of excess fatty acid in the reaction mixture so that it does not reach more than 10 g/l.
• the fatty acid levels are suppressed by adjusting the level of substrate fed to the metabolizing cells. For example, on the first day after inoculation, starting immediately after inoculation 0.5 g/l.h of oil is added, after that 1.5 g/l.h of oil is added unless the fatty acid level becomes too high.
• the fatty acid level of the reaction mixture is suppressed below 10 g/l, preferably below 8 g/l, more preferably between 1-5 g/l, expressed in weight of acid per liter of reaction mixture.
• Suppression of the fatty acid level has for effect that the resulting mixture of sophorolipids formed is less complex. It has the advantage that conditions for the crystallization of sophorolactone are created. Access to this compound in solid form, allows the lactone and acid form to be separated without the use of organic solvents. It has the further advantage of yielding very low levels of residual fatty acid in the sophorolactone composition.
• Formation of fatty acid in the reaction mixture must be suppressed below a predetermined level such that at least part of the sophorolactone produced will become available in the form of crystals.
• the reaction mixture Upon production of a sufficiently high yield of the desired compounds, the reaction mixture is warmed to a temperature between 60°C and 90°C, preferably 70 °C, thereby melting the sophorolactone crystals.
• the molten sophorolactone is allowed to settle to the bottom of the fermenter.
• a lower layer will form providing a crude sophorolactone composition.
• the crude sophorolactone composition can be removed from the remainder of the reaction mixture without difficulties.
• the addition of an organic solvent, e.g. to extract the sophorolactone from the mixture is not required. This is advantageous as it avoids that the resulting sophorolactone is contaminated with traces of organic solvent. Because of the high temperatures and exposure time this process will have the effect of pasteurization. This results in killing off the yeast cells thus facilitating disposal of the broth remaining after product extraction.
• the filtration step removes cell debris from the sophorolactone composition.
• the process comprises the following purification procedure. This procedure can be carried out on the crude sophorolactone composition which has settled to the bottom of the fermenter. It can also be carried out on a crude sophorolactone composition which has been removed from the fermenter.
• the crude sophorolactone composition is cooled to a temperature whereby the sophorolactone solidifies.
• the composition is cooled preferably until a paste is formed . If the paste is still in the fermenter it is drained from the fermenter. For example, by opening the fermenter at the bottom and draining the crude sophorolactone composition from the fermenter. Alternatively the supernatant broth is removed and the paste is allowed to be formed in the fermenter. Formation of a paste can be improved by seeding with a dry lactone powder material from a previous production.
• the solid such as a paste
• the solid is dispersed in water at a temperature of 5-25 °C, preferably 10-15 °C; more preferably at 10 °C.
• the mass ratio of water to solid is preferably between 0.5 and 15.
• the use of this amount of water assures that sophorolipids present in the acid form dissolve well in contrast to the sophorolipids present in lactone form. These are poorly water soluble. The poor water solubility is even worsened by the temperature of the water. The sophorolactone present crystallizes and an aqueous dispersion of sophorolactone results.
• decalcified water is used.
• Calcium ions present in the water would otherwise form calcium salts with the sophorolipids present in acid form.
• the formation of calcium salts is undesirable because it hampers the valorization of sophorolipids other than the sophorolactones.
• the sophorolactone is separated from the water; e.g. by decantation or centrifugation. This has for effect that the sophorolipids present in the dispersion are fractionated.
• the acid form remains in the water, whereas the sophorolipids in lactone form, i.e. sophorolactone, is separated off, in the form of crystals.
• the procedure of dispersing and separating may be optionally repeated. Preferably the steps are repeated 0 to 10 times.
• the temperature of the water is kept below 40 °C, preferably below 30 °C during the procedure. This has for effect that the crystals are not molten. Keeping the sophorolactones in crystal form has the advantage that optimum separation between the acid and lactone form results and the amount of water in the sophorolactone is reduced to a minimum.
• the crude sophorolactone composition is brought to a temperature between 35 °C and 55 °C to melt the sophorolactone crystals present, thereby providing a lower layer comprising sophorolactone and an upper layer of supernatant water; separating the lower layer from the upper layer to obtain a composition with increased sophorolipid content versus the crude sophorolactone composition.
• the resulting sophorolactone composition i.e. paste or crystals
• the crystals can be dried initially by centrifugation. Additional drying techniques can be used to dry the crystals further.
• the purification and drying step results in a sophorolactone in solid form. In a preferred embodiment the drying is by spray drying, drum drying, convection drying, thin film evaporation, vacuum drying, flaking, extruding or casting.
• the resulting supernatant water fraction is preferably recycled.
• the first volume of wash water contains sufficient active matter for it to be used economically.
• subsequent washing steps are necessary to obtain a white end product. This will yield wash water with lower amounts of sophorolipids than in a preceding wash step, which would be regarded as waste.
• Step 1 provides a washing slurry from the raw sophorolipids (i.e. the bioconversion product) and the wash water obtained from step 2 (as stored from a previous purification run). This slurry is then separated into semi- washed lactone and wash water rich in sophorolactone. The semi-washed lactone is then suspended again, this being step 2, in wash water from a previous step 3 and so forth.
• Step 5 does not use wash water, but fresh water instead.
• wash water Preferably two volumes of wash water are used for each volume of raw sophorolipids.
• the washing water travels from the end of the sequence back to the front, accumulating the water soluble components, and the insoluble lactone travels in the opposite direction.
• the resulting process results in a counter current purification of the sophorolactone with reduced amounts of waste and fresh water requirements. Due to the selectivity of a process according to an embodiment of the invention, problems with foam and the need for foam suppression are reduced.
• problems with foam and the need for foam suppression are reduced.
• small amounts of yeast and cell fragments from the micro-organism may become entrapped in the drained precipitate.
• a border layer of yeast cells on top of the warm sophorolipids is formed. Part of this layer may also be drained along with the sophorolipids, both sources resulting in small amounts of particulates in the products.
• the inventors were able to solve the problem of entrapped material by the use of depth filtration.
• the process additionally comprising the step of filtering the sophorolactone separated off the reaction mixture by depth filtration .
• depth filtration is carried out at a temperature between 40 °C and 90 °C and a pressure of between 0.5 bar and 2.5 bar.
• Most preferably depth filtration is carried out at a temperature between 60 °C and 70 °C and a pressure between 0.5 and 2.0 bar.
• the sophorolactone separated composition off the reaction mixture is in the form of an aqueous sophorolipid mixture comprising at least 30 wt%, preferably at least 70 wt%; more preferably at least 80 wt% sophorolactone, expressed on the dry matter content of the aqueous sophorolipid mixture.
• the sophorolactone is comprised in a composition comprising at most 60 weight percent water; preferably at most 50 weight percent water; most preferably at most 40 weight percent water.
• the fermentation is executed in a vessel at an overpressure of between 0-1.5 bar, preferably at 0.5-0.6 bar. This has the advantageous that foam formation can be suppressed.
• the present invention provides a sophorolactone composition in the form of a solid or paste, obtainable by a method according to an embodiment of the invention, consisting of 15-70 wt% sophorolactone, preferably 30-70 wt%; 0-30 wt% sophorolipid acid, preferably 0- 15 wt%; 0-1 wt% fatty acid, preferably 0-0.5 wt%, and remainder water.
• sophorolactone composition obtainable by a method according to the invention comprises at least 60 weight percent sophorolactone and at most 40 weight percent water.
• the sophorolactone composition consists of 70 - 100 wt% sophorolipid lactone, 0 - 1 wt% sophorolipid acid, preferably 0-0.5%, less than 0.1 % fatty acid, and remainder water.
• the sophorolactone composition consists of at least 95 wt%, more preferably at least 98 wt%, most preferably at least 99 wt% sophorolactone.
• sophorolactone resulting from a method of the invention which is essentially free of water, can advantageously be combined with these solvents to provide a storage stable sophorolactone composition.
• the present invention provides a formulation obtained by dissolving the sophorolactone composition in a solvent which dissolves sophorolactone particularly well.
• the inventors have found a range of solvents that are particularly suitable for dissolving sophorolactones. These solvents have in common that they can be characterized by similar Hansen Solubility Parameters. Each solvent investigated was given three Hansen parameters, each measured in MPa : delta D characterizing the energy from dispersion bonds between molecules, delta P characterizing the energy from dipolar intermolecular forces between molecules and delta H characterizing the energy from hydrogen bonds between molecules. The results are summarized in a graph in Figure 1 and in Table 2. In Figure 1, the datapoint numbers representing the percent sophorolactone dissolved at room temperature by the solvents with the particular delta P-delta H combination.
• the 27 solvents tested in the experiment and depicted in the Figure are as follows : water, glycerol, 1.3 propanediol, propylene glycol, caprylyl glycol, ethanol, phenoxyethanol, benzylalcohol, methanol, isopropanol, phenetyl alcohol, glycerin formal , ethylacetate, DPM, PNP, PNB, ethyllactate, isoamyl lactate, butyl lactate, glacial acetic, D-limonene, hexanol, dimethyl lactamide, DMI, glycerincarbonate, 2-3-o-isopropyliden glycerin, hydrocinnamylalcohol.
• sophorolactone in excess of 15% : phenoxyethanol, benzylalcohol, phenetyl alcohol, glycerin formal, DPM, PNP, ethyllactate, isoamyl lactate, butyl lactate, dimethyl lactamide, DMI, 2-3-o-isopropyliden glycerin, hydrocinnamylalcohol.
• the solvent in a formulation according to an embodiment of the invention is selected from the list of phenoxyethanol, benzylalcohol, phenetyl alcohol, hydrocinnamylalcohol, tetrahydrofurfuryl alcohol, dimethylisosorbide, methyl salicylate eugenol, linalool, hexanol, glacial acetic, dimethylcarbonate, certain glycolethers such as dipropyleneglycol methyl ether and 1-propoxy 2-propanol and lactate esters including ethyl-, butyl-, amyl-, ethylhexyl-lactate.
• the solvent in a formulation according to an embodiment of the invention has a dispersion parameter in excess of 17 (MPa) 1/2 .
• solvents include for example phenoxyethanol, benzylalcohol, phenetyl alcohol, hydrocinnamylalcohol, tetrahydrofurfuryl alcohol, dimethylisosorbide, methyl salicylate and eugenol.
• the method of the invention provides an economically interesting access to sophorolactone with improved purity. In turn, this opens up new possibilities.
• a method according to an embodiment of the invention provides sophorolactone in dry form.
• the virtual absence of water makes it possible to use it as auxiliary in formulations with alkaline pH, such as alkaline washing powders and washing tablets.
• the sophorolactone hydrolyzes to its acid form, causing the formulation to change its foaming behavior.
• the foaming behavior of the formulation remains stable. The application requires low foaming behavior. Increased foaming could lead to blockage of the automated dishwasher wherein these tablets or powders are used.
• the invention provides a dry solid product for laundry or dishwashing, comprising the sophorolactone composition consisting of 70 - 100 wt% sophorolactone, 0 - 1 wt% sophorolipid acid, preferably 0-0.5% sophorolipid acid, less than 0.1 % fatty acid, and remainder water, wherein the pH of the product is 8.0-14.0, preferably 8.5- 11.50, as measured on a 1% solution of the product in water.
• the invention provides a process for the preparation of partially hydrolysed sophorolactone.
• a sophorolactone obtained by a process according to the invention is used. At least part of the sophorolactone is hydrolysed .
• sophorolactone is added to the sophorolactone. Between 0.5 to 1 equivalents of base are used compared to the sophorolactone.
• Partially hydrolysed sophorolactone compositions thus obtained have the advantage that their high purity is maintained and at the same time the low water solubility of the formulation is improved by providing lactone in acid form which solubilizes the lacton.
• sophorolactone is partially hydrolyzed, providing a formulation whereby of the sophorolipids present 39-45% are in the acid form and 55%-61% are in the lactone form.
• hydrolysis of at least part of the sophorolactone is obtained by heating the sophorolactone.
• the sophorolactone is heated for more than 4 hours, more preferably for more than 24 hours.
• the sophorolactone is heated to a temperature between 70-90°C, preferably to a temperature between 80-90 °C.
• sophorolipid lactone which is at least partly hydrolysed to sophorolipid acid and corresponding salt.
• the advantage of this method is that a composition with pre-determined foaming behavior can be obtained. Foaming behavior can be changed from non-foaming corresponding to a composition comprising 100% sophorolactone to a composition which is highly foaming comprising 100% hydrolysed sophorolactone or sophorolipid in acid form. As a sophorolactone was used that is substantially free of water, the resulting composition can also be obtained as substantially water free. Hence, an composition with improved hydrolytic stability is obtained. The foaming behavior remains stable during storage.
• the invention further provides a method for the preparation of ⁇ hydroxy fatty acids of formula CH20H-CH2-(CH2)n-COOH (IV) or ⁇ -l hydroxy fatty acids of formula CH3- CHOH-(CH2)n-COOH (V).
• These acids are obtained by starting from a sophorolactone obtainable using a method according to an embodiment of the invention.
• the sophorolactone is hydrolysed to the corresponding diacetylated sophorolipid in acid form, preferably by hydrolysing the sophorolactone using a base.
• the sophorolipid obtained is submitted to ozonolysis, removing any salts and dicarboxylic acids in order to obtain sophorolipids with a chain length of 9 carbon atoms.
• the head and tail portion of the molecule are separated. Separation can be obtained by hydrolysis with a weak acid or by enzymatic reaction. This results in the sugar moiety of the molecule and the ⁇ and ⁇ - 1 hydroxy fatty acid moiety.
• the sugar moiety and/or hydroxy fatty acid are recovered. Preferably both the sugar moiety and hydroxy fatty acid are recovered.
• a sugar moiety corresponding to sophorose is of great economic value. It is a potent inducer of cellulase production e.g . by the fungal species Trichoderma rees/V.Cellulases are widely used ie.g. in laundry washing and textile seizing, and production of bioethanol from cellulosic material.
• Trichoderma rees/V.Cellulases are widely used ie.g. in laundry washing and textile seizing, and production of bioethanol from cellulosic material.
• the process of the invention provides access to a route which starts from sophorolactone with improved purity. This facilitates the access to a purer sophorose. As the sophorolactone production can be practised on a large scale, larger scale sophorose production becomes accessible.
• Sophorose obtained by a method according to the invention may be used as inducer of cellulase producing micro- organism e.g. Trichoderma .
• Sophorose may be used as inducer for the enzyme cellulase, preferably for use in the production of ethanol.
• the sophorose obtained by the method of the invention provides access to a lower cost carbon source for the fermentation of sugars by cellulase thereby providing a more interesting access route to bio-ethanol.
• sophorolipids are split into sophorose and hydroxyl fatty acids.
• sophorose is destroyed as a strong acid hydrolysis is used.
• sophorose valorization is advantageous. It is e.g.
• the ⁇ and ⁇ -l hydroxy fatty acids obtained by a method according to the invention have the advantage that they are derived from a process wherein the level of fatty acids of formula ROH wherein R is an alkyl chain, was kept very low.
• the resulting ⁇ and ⁇ - 1 hydroxy fatty acids are characterized by lower levels of non-hydroxylated fatty acids of than in prior art methods, in particular below 0.8%. This is advantageous as this impurity is known for its ability to terminated polymerization reactions.
• the process of the invention results in product which is better suitable for polymerization reactions due to the substantial absence of these polymerization reaction terminators.
• this preculture is essentially free of oily substrate and it was used in the stationary rather than in the exponential growth phase.
• the preculture is used to inoculate a previously sterilized Biostat U50 (Sartorius BBI Sytems) containing 40 liter sterile medium with the same composition as the preculture.
• a double fedbatch fermentation was operated at 110-130 rotations per minute (rpm), 25°C, 0.5 bar and pH 3.5.
• Aeration was set at 1 vvm (amount of the introduced air versus the bioreactor's working volume), decreased to 0.1 vvm after 40 hours in order to control excessive foam formation, and raised again after 180 hours to 1.3 vvm.
• rapeseed oil Bioplanet, food grade
• the continuous feeding rate was set at 1 g/l.h.
• the feeding rate was controlled as a function of the residual oil and the residual oleic acid, targeting an oleic acid content of 1-5 g/l, at all times avoiding concentrations above 8 g/l.
• the feeding profile is illustrated in Figure 2.
• the presence of residual oil in the fermentation broth was measured by taking a sample of fermentation broth, heating to 80°C, and by visual inspection observing if a layer of oil is formed. Solid glucose was added as soon as the concentration dropped below 20 g/l.
• the fermenter was then heated to 90°C in order for the crystals to melt and to obtain an oily layer which after cooling is easily separated from the broth by draining the fermenter the next morning. Once stored in drums the sophorolipids readily crystallized again to take the shape of a hard solid.
• Example 1 The experiment of Example 1 was repeated this time adding 5 g/l methyloleate (Cargill) to the preculture and continuously feeding methyloleate instead of rapeseed and feeding a total of 240 g/l to the fermenter.
• the ester feeding profile was again adapted to a maximum concentration of 5 g/l oleic acid.
• the preculture was grown for 24 hours at 25°C. It was in the exponential growth phase when used for seeding.
• a total sophorolipid yield of 262 g/l was obtained after 222 hours, with a production rate of 1.18 g/l. h and a substrate conversion of 0.58 (cf. Table 3).
• Several lactonic peaks as well as approximately twice the amount of acid sophorolipids are observed.
• Example 2 The experiment of Example 2 was repeated with rapeseed instead of methyloleate.
• the preculture was grown for 30 hours at 25°C and was in the exponential growth phase when used for seeding.
• the medium of both the preculture and the fermentation broth was made according to Lang et al (2000).
• Five gram per liter rapeseed oil (Bioplanet, food grade) were added to the seeded preculture.
• a total of 240 g/l rapeseed was fed to the fermenter.
• a feeding profile was adapted targeting a maximum concentration of 5 g/l oleic acid.
• a total sophorolipid yield of 320 g/l was obtained after 240 hours, with a production rate of 1.33 g/l.h and a substrate conversion of 0.8 (cf. Table 2).
• An HPLC analysis revealed a clear peak at 24.4' indicating the presence of acid sophorolipids. These were probably the cause no crystallization occurred. Only upon storage for several weeks at 4°C did gradual crystallization occur.
• Example 1 was repeated in a Biostat D100 containing 70 liter of CSL medium at 25°C to which a total of 240 g/l rapeseed oil was added controlling the residual oleic acid content.
• the fermenter was inoculated using 1.4 liter CSL medium without oil and grown in 7 1- liter Erlenmeyers for 72 hours at 25°C until the stationary phase was reached. After 183 hours 254 g/l sophorolipids were present again virtually free of the acid form as evidenced under the microscope by the presence of crystals during the fermentation.
• the production rate and the substrate conversion were respectively 1.39 g/l.h and 0.62%.
• Example 5 Example 5
• Examples 4 and 5 confirm the importance of an oil-free preculture in the stationary phase for obtaining crystalline sophorolipids rich in the lactone form.
• Example 4 was repeated on a D 100 fermenter feeding 240 g/l rapeseed oil in total to 70 liter of CSL medium, but this time working with 3 liter preculture cultivated in a Biostat B5 rather than Erlenmeyers. Four fermentations were set-up in the D100 varying the nature of the preculture.
• the FB23 and FB35 precultures did not contain rapeseed oil, FB32 and FB34 contained 5 g/l rapeseed oil.
• the precultures were grown for a varying period of time until they reached a varying point of the stationary phase as summarized in Table 5 along with the results.
• a large scale production of sophorolipids was performed according to the method of the invention.
• a double fedbatch fermentation was performed in 3 stages:
• Preculture 1 consisted of 2 baffled Erienmeyer flasks of 2L with 0,5 L CSL medium as described in Example 1, but with only 55 g/l glucose rather than 110 g/l. After the sterilization, each Erienmeyer was inoculated with 1 glycerol vial (2 mL in a Cryovial). The Erlenmeyers were shaken at 25°C. After 45 hours of growth, the culture reached the mid- exponential growth phase (Optical Density between 25 and 30) . Inoculation in 100 L medium was carried out after 49 hours of growth.
• Preculture 2 consisted of 100L CSL medium as described in Example 1. Preculture 2 was grown in a 150L fermenter. All media components were dissolved in decalcified water and before sterilization 10 mL of an antifoaming agent (Entschaeumer A 4050 HAC) was added. The pH of the medium was 5,7 which was not corrected for. All components were sterilized together for 35 minutes at 121°C. After sterilization, the pH reached a value of 5,4. After sterilization, the fermenter was inoculated with the Erienmeyer preculture (two Erienmeyer flasks with 0,5L culture). At the moment of inoculation, the Erienmeyer preculture had grown for 49 hours and reached an optical density of 21,8.
• an antifoaming agent Entschaeumer A 4050 HAC
• the glucose in the Erienmeyer Preculture was not depleted at that moment and the pH was approximately 3.
• the Fermenter Preculture was grown at approximately 25°C, the temperature varied between 22 and 27°C. Oxygen limitation was avoided by fully opening the valve of the incoming air. An overpressure in the fermenter of 20 kPa was performed.
• the main fermentation was carried out in a 8 m 3 bioreactor with a working volume of 6,5 m 3 .
• the fermentation started with a volume of 3,5 m 3 corn steep liquor (CSL) medium.
• CSL corn steep liquor
• 1,75 kg rapeseed oil (amount for 1 hour) was added to the medium before sterilization. This gave time to install the pump for the feeding of the rapeseed oil and to connect all necessary tubings after the inoculation. So, fedbatch of the oil was started 1 hour after the inoculation.
• the fermenter was inoculated with the fermenter preculture ( 100L culture). At the moment of inoculation, the fermenter preculture was grown for 72 hours and reached an optical density of 51. The glucose in the preculture was not depleted at that moment (tested with glucose strips available from Roche) and the pH was approximately 3,5.
• the fedbatch fermentation was performed at approximately 25°C, the temperature varied between 24 and 27°C. After 24 hours of fermentation, cooling became a problem because of the enlarged heat production of the culture (exponential growth phase). An extra cooling pump was switched on in order to keep the temperature at 25°C.
• the airflow was set at 1,5 m 3 /min.
• An overpressure in the fermenter of 500 kPa was performed.
• the agitation was approximately 75 rpm. Due to foam formation, which started after 36 hours of fermentation, the airflow had to be decreased several times in order to control the foam formation.
• the minimal air flow was 0,4 m 3 /min.
• the pH decreased to 3,5 and was hereafter kept constant at this value by adding 10 N NaOH by means of a piston pump.
• the pH started to decrease after 4 hours and reached 3,5 after 11, 5h of fermentation.
• the pH varied between 3,4 and 3,9 as a consequence of the addition of the NaOH.
• Approximately 80L of 10M NaOH were added. Most of NaOH was added during the first 48 hours of growth.
• extra rapeseed oil and glucose were added.
• Rapeseed oil was added non-sterile as a continuous flow. A total amount of 240 g/L (in relation to the starting volume) was added. This means a total volume of 916 L or 840 kg. The feeding rate of rapeseed was adjusted in function of the residual fatty acid content. This content was determined by extraction of a fermentation broth sample with hexane. This was done in such a manner as to not exceed a residual fatty acid content of 5 g/l and not to observe any residual oil on top of a heated sample after day 2. A total amount of 798 kg or 228 g/L of rapeseed oil, expressed in relation to the starting volume, was added to the culture.
• Solid glucose was added discontinuously by opening the top plate after pressure release because there was a low risk for contamination during the fermentation. Addition was function of the actual glucose concentration in the culture, such that as soon as the concentration dropped below 20g/l glucose was added. A total amount of 1250 kg glucose. H 2 0 was added during the fermentation.
• sophorolipids were collected from the bottom of the fermenter. It was necessary to steam the wall of the fermenter, because re-crystallization had occurred at the (cold) wall of the fermenter. Two barrels of 640L and 9 barrels of 50L were filled with sophorolipids. Only the last barrel contained impure sophorolipids mixed with yeast cells and cell fragments. The sophorolipid fraction drained from the fermenter was in the form of a paste.
• Sophorolipids were obtained as a liquid from the aforementioned fermentation of Example 7 through decantation. This dark brown liquid is allowed to solidify and will be referred to as "sophorolipid paste". It contained approximately 50% water and 0.1% cell fragments (visible as a haze when dissolving the paste in ethanol). The lactone form solidified at 30- 35°C resulting in a steep increase of the sophorolipid fraction. An amount of paste was dispersed in roughly the same weight of cold tap water to observe an almost immediate formation of an creamy opaque dispersion of soft white thin sharp lactone crystals of approximately 1-3 micron wide and 20-50 micron long with a crystal density of about 1.070 kg/I. The crystal phase accounted for approximately 30% of the dispersion volume.
• the first wash water contained approximately 20-25% dry matter with the HPLC profile as depicted in Figure 3.
• Example 8 was repeated in 25 liter canisters. The lactone fraction was allowed to settle overnight. A clear supernatant was decanted. It was replaced with the same amount of fresh tap water, redispersed and again allowed to settle. The brown color gradually disappeared. After 10 cycles a pure white lactone fraction was obtained with the HPLC profile as depicted in Figure 4.
• sophorolipid paste from Example 7 was homogenized for 5 minutes in order to avoid undispersable lumps forming in the dispersion.
• the homogenized paste was dispersed in 4400g of decalcified water through vigorous mixing, until a homogenous dispersion was obtained whilst cooling at 10°C.
• This dispersion had a liquid density of 1039 kg/I and a viscosity of 5,8 cP.
• a total of 6000g of the initial dispersion was centrifuged (speed : 4000rpm, radius: 190mm, duration : 10 minutes).
• Example 10 was repeated, with a total of five cycles of adding water to a paste of sophorolactones, mixing to obtain a dispersion and subsequently removing the bulk of the watery phase through centrifugation.
• 1190 gram of sophorolipid paste was dispersed in 1800 gram of cold water.
• the lactone fraction was collected after centrifugation and decantation. All five liquid phases which were removed by centrifugation were analyzed for their respective dry matter content. This allowed for the total dry mass in the original paste to be determined, as well as the amount of water- soluble components.
• Table 6 displays these results. As can be seen from Table 6, the original fermentation product contains approximately 57% dry matter. Directly determining the dry matter content of the fermentation product led to an overestimation of this value.
• the method used to determine these values was an automated infrared balance which, for the original product gave values for dry matter of 61,3% (at 80°C) and 60,2% (at 140°C).
• this paste was diluted according to the first step of the process, a value of 57,3% (at 140°C, after correcting for the dilution) was obtained, this being closer to the total amount of dry mass recovered during the separation .
• the original paste itself is probably too hygroscopic and/or bulky to effectively release all of its water. When, through diluting, less actual dry matter is analyzed, it is expected that comparatively more water would evaporate and a more accurate reading is obtained.
• the dispersion consists of the solid, water insoluble, lactone and an aqueous phase with solvated lactone, water soluble partially acetylated lactone and sophorolipids in acid form as well as various salts, sugars and colored components. Approximately 30% can be separated as insoluble lactonic sophorolipids, i.e. sophorolactones. The remaining 70% are almost all to be found in the first three wash liquors, roughly distributed as 80/17/3. This remaining 70% are both water-soluble components as well as water-insoluble lactone as evidenced earlier by HPLC in Example 8.
• wash water is a terna ry system consisting of water, water-soluble components (acid sophorolipids and other amphiphilic components in the medium) that solubilize the insoluble lactone and the lactone itself, with the ratio between the latter two remaining constant irrespective of the amount of water.
• a sample of 171 g sophorolipid paste was dispersed in 342 g water as described previously, the sophorolipid concentration of 333 g/l approximating that of a typical fermentation broth .
• This dispersion contained 89 gram sophorolipids of which 26 g could be isolated as lactone.
• 0.45 gram up to 10.7 gram was added of a fatty acid mixture ("FA" in Table 8 below, Radiacid 0223 of vegetable origin, similar in composition as the vegetable oil used for feeding) to observe the lactone separation and the characteristics of the supernatant.
• the dispersion was vigorously stirred and an additional amount of fatty acids was added (e.g. another 0.45 g to obtain the second row in the table below, another 0.89 g for the third row, etc).
• the minimal relative lactone content of a sophorolipid mixture has to be greater than 30% by weight in order for the successful removal of lactonic sophorolipids by dispersion in water. Such was determined by saturating a mixture of water-soluble sophorolipids with lactonic sophorolipids. The watery phase was then analyzed by reverse phase high performance chromatography and it was found that the lactone sophorolipid content of the watery phase increased to a value of 30% by weight, relative to the total sophorolipid amount. Therefore it follows that any sophorolipid mixture containing an amount of lactone form, this form must be present in excess of 30% by weight expressed relative to the total amount of sophorolipids, in order for a portion of this lactone form to precipitate as a solid.
• the purer lactonic sophorolipids liquefied and settled to form a separate phase at temperatures in excess of 45 °C.
• the separate phase was isolated. After isolation it was transferred to storage, especially for large volumes, or left to solidify for easy removal of the small volume of supernatant watery phase. Afterwards, the lactone was obtained as a white solid which contained approximately 15% of water. Analysis of the watery phase indicated that less than 1% of the lactone remained dispersed. Further air drying of such material and grinding resulted in a non-sticky powder with a melting point of 79°C.
• the inventors have found that the small yeast fragments can be removed by dead-end filtration. This filtration is suitable for use on the crude sophorolactone composition in the liquid state drained from the fermenter.
• the lactone obtained in Example 10 was partially hydrolysed in degrees ranging from 0 to
• 500 g crude mix contains 92 g insoluble lactone or with a MW of 689 this corresponds to 0.13 mol.
• a 0.7 equivalents partial hydrolysate thus requires 0.7*0.13 mol KOH or 0.7*0.13*56.11 g KOHi 00 % or 10.5 g KOH 50 %.
• 10.5 g KOH 50 % and 489.5 g water was added to the 500 g crude mix. This mixture was homogenized and allowed to react at 80°C for 4 hours, 60°C for approximately 6 hours, or for 3 days at 45°C until a clear solution was obtained with a pH of 6.5 or less, the pH typically stabilized at 5.5 and a clear liquid layer settled from the reaction mixture.
• the mixture now contained sufficient water soluble sophorolipids, i.e. sophorolipids in acid form, to solubilize the remaining lactone in a separate aqueous phase.
• Table 10 summarizes the data for all hydrolysates. From the results in Table 10 it follows that the double acetylated lactone form gradually disappears upon partial hydrolysis and from 0.8 equivalents onwards some fully hydrolysed material was present.
• a sophorolipid lactone sample obtained from the experiment described in Example 8 was centrifuged, spread out on Torek paper and allowed to air dry. The resulting solid was crushed with a coffee mill to a fine powder and dried further with compressed air to >98 wt%. This powder was compared as to its efficiency in automatic dishwashing versus a low foaming alkylpolyglucoside, in particular Simulsol AS48, available from Seppic SA, at equal active matter content.
• the surfactant was formulated in the following base formula : 9.43% sodium citrate.2H 2 0; 3.08% percarbonate; 2.72% sodium carbonate; 2.05% sodium silicate; 1.05% sodium bicarbonate; 0.77% sodium polyaspartate; 0.62% tetra acetyl ethylene diamine (TAED) + 1.4 % surfactant.
• 20 gram of the base formula was dosed in a ZDF211 dishwasher. Program D 50°C eco was run. In the automatic dishwasher 7 types of soil plates (available from CFT, Vlaardingen, The Netherlands) mounted in a stainless steel holding frame were inserted. The results obtained are summarized in Table 12.
• Table 12 Stain removal efficacy for Simulsol AS48 compared to lactone for a series of stains.
• sophorolipid lactone although virtually insoluble as such, was superior to Simulsol AS48. It is assumed the alkaline pH of about 9.8 caused in situ hydrolysis providing an efficient surfactant.
• a stock solution was prepared containing 7.5% partially hydrolysed sophorolipids, 5% surfactant (Glucopon 215), 3% ethanol and 0.5% glycerol monocaprylate.
• the pH of the formulation was set at 6.3 using citrate and lactic acid.
• Table 13 Storage stability data of samples with varying levels of oleic acid, measured at different temperatures.
• Sophorolactone compositions as obtained in Examples 1 to 7 or sophorolactone as obtained in Examples 8 to 15 were completely hydrolysed by adding 3,2 equivalents KOH and water to obtain a 10 % dry matter solution.
• the reaction mixture was heated to 50 °C, stirred for 10 minutes and passed twice over a glass fiber filter.
• the mixture was heated to 80°C during 4 hours to complete the hydrolysis and obtain the deacetylated acid sophorolipid.
• the pH of the mixture was adjusted to pH 1.5 using 37% HCI and left to precipitate overnight.
• the acid sophorolipids were separated from the supernatant salt solution (KCI and potassium acetate) by centrifugation.
• the sophorolipids were washed and redispersed twice with 1% HCI, collected by centrifugation, air dried and ground to a white powder.
• Residual substrate determination was advantageously carried out as follows: a sophorolactone sample of 200 g was liquefied by warming it to 60°C. It was then extracted twice with an equal volume of n-hexane in a separation funnel. Subsequent evaporation of the pooled extracts yielded the bulk of residual free fatty acids. The residual free fatty acid level was then determined by weighing the amount of residue obtained and calculation of the amount obtained relative to the original sample weight. Having the sophorolipids in a liquid phase greatly increased the speed of phase separation from about one day, using solid sophorolactone as starting material, to half an hour, using the liquefied material. It was also found that using cyclohexane instead of n-hexane resulted in a co-extraction of an unacceptable amount of sophorolactone, where n-hexane did not.
• Example 21 effect of too high level of residual free fatty acid level in fermentation broth
• Three flasks containing 1500 ml pre culture medium were each inoculated with Candida bombicola. After 72 h incubation at 25 °C the content of the three flasks was pooled for inoculation of the seed tank. The development and morphology of the inoculum shake flasks was in line with the results of the lab scale experiments. Limited foaming was observed till approximately 36 hours. No foreign growth was detected.
• the seed fermentation was inoculated by approximately 4.5 I, 72 h old, shake flasks material. After a somewhat slow start, the seed tank developed well. After 66 hours, still in the stationary phase, approx.
• the main fermentation was inoculated by approximately 1 m 3 of the 2.2 m 3 seed tank material, of 66 hours of age. During the process non-sterile rape seed oil was continuously fed whereas glucose was added in shots. Within the window of 198-216 hours for operational reasons, the main fermentation was terminated at 204 hours after inoculation . The broth weight at this time was determined at approximately 41 tons, 31% by volume of which was a sophorolipid mixture settling upon heating.
• the dosing of glucose was based on the actual residual glucose concentration of the broth, which during the total course of fermentation did not drop below 60 g/l. Divided over 5 shots 5639 kg pure glucose was dosed. Together with a batch dosing of 2593 kg in total 8232 kg of glucose was added to the process.
• the rape seed oil feeding was not controlled based on the maximum allowable level of 10 g/l fatty acid content of the broth. Feeding started directly after inoculation, in total 6300 kg of rape seed oil was added. Eight hours before the end of fermentation the rape seed oil feed was stopped. The material obtained stayed liquid. Sophorolactone could not be selectively retrieved from the reaction broth without use of organic solvents.
• Raw sophorolactone from a method according to an embodiment of the invention typically results in raw sophorolactone with a water content of 35-40%.
• Example 22 storage stability test
• a sample was prepared of 10 g sophorolactone and 90 g ethyllactate and another sample of 10 g sophorolactone and 90 g isoamyl lactate. Both samples were split in 50 ml samples and stored at 4°C and at 40°C respectively. After 2 months of storage they were analyzed with HPLC-ELSD. The results are as illustrated by the chromatograms depicted in Figure 7 and Figure 8. After 2 months of storage at either 4 °C, result depicted in Figure 7, or 40°C, result depicted in Figure 8, of this non-aqueous sophorolactone solution only a negligible hydrolysis of lactone material occurred as evidenced by an acid sophorolipid peak appearing at 30,5 min in the grey overlay graph . This peak represents less than 1% of the total peak area.

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